Grinding samples using rotational and linear motion

ABSTRACT

The disclosure is directed to a sample preparation apparatus for grinding or homogenizing test samples. More specifically, the disclosure relates to grinding samples using rotational and linear motion. Grinding samples can be accomplished with an apparatus with a slider-crank mechanism that is attached to an oscillating connecting linkage. The amplitude of oscillatory motion can be greater than or equal to a length of a sample processing chamber.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. Application No. 16/710,979 filed Dec. 11,2019 and entitled “Methods for Grinding of Samples Using a Combinationof Rotational and Linear Motion,” which is a divisional of U.S.Application No. 15/702,609 filed Sep. 12, 2017 and entitled “Methods ForGrinding of Samples Using a Combination of Rotational and Linear Motion”(now U.S. Pat. No. 10,539,486 issued Jan. 21, 2020), which is acontinuation of U.S. Application No. 14/257,632 filed Apr. 21, 2014 andentitled “Apparatus and Method for Grinding of Samples for Analysis”(now U.S. Pat. No. 9,759,638 issued Sep. 12, 2017), which claims thebenefit of priority to U.S. Provisional Application No. 61/816,094 filedApr. 25, 2013 and entitled “Mechanism for Grinding of BiologicalSamples,” each of which is expressly incorporated by reference herein inits entirety for all purposes.

BACKGROUND Field

The present invention is directed to hand held and table top grindersand homogenizers such as those used to release proteins and nucleicacids from biological samples and those that grind geological samplesfor composition analysis. Samples may include, for example, seeds, soil,bones, teeth, tissues, spores, yeasts, and gram positive microbes.Certain embodiments, described below, result in more efficient andfaster processing of said samples.

The most commonly used homogenizers are of the bead mill type(bead-beater), in which a biological or geological sample is added to acontainer such as a sample vial or tube that is preloaded with grindingmedia such as, but not limited to, for example, ceramic, glass or metalbeads. A special buffer designed to dissolve proteins, nucleic acids,minerals and/or metals may also be added, followed by sealing of thetube typically with a screw cap. The sealed tube is then placed into thehomogenizer, which mechanically agitates it in an oscillatory manner,causing the media (hard beads) within the tube to impact and break thesample. Typical rates of oscillation are in the range of 4,000 to 5,000cycles per minute. This grinding or homogenization of the samplefacilitates the extraction of proteins, nucleic acids, metals and/orminerals for use in downstream processes such as amplification, analysisof DNA and/or analysis of metal/mineral composition.

The biggest drawback with most bead mill homogenizers is that theirrange of oscillatory motion is not high enough to cause the beads totraverse the whole length of the sample processing chamber such as asample vial or tube. Furthermore, if the sample tube is orientedvertically in the homogenizer, gravity tends to cause the beads tolocalize at the bottom of the tube during homogenization. At the sametime, the biological sample, which is less dense than the beads willtend to stay closer to the top of the tube. As a result, the time toachieve complete homogenization is long, usually in the range from 5 to60 seconds; perhaps longer if the sample is also hard (e.g. dry cornkernel, bone, etc.). Another undesired effect from this inefficienthomogenization scheme is the generation of heat proportional to thelength of time of homogenization due to internal friction within thetube. This heat is problematic if the desired nucleic acids are RNAs,which quickly degrade at elevated temperatures.

The previously mentioned drawbacks could be mitigated if the amplitudeof oscillatory motion were to be extended to be at least equal to thelength of the tube being used to process a biological sample such asthat in the apparatus of the present invention. Doing so would cause themilling beads within the tube to traverse the whole length of the tube,enhancing the incidence of encounters between the beads and the sample.This more efficient design of the apparatus of the present invention inturn results in shorter homogenization times and thus, less heatgeneration. The preferred embodiment of the present invention hassubstantially improved grinding/homogenization efficiency, leading toshorter homogenization times, and less heat generation compared to thosein the prior art.

Description of Related Art

There are a number of bead mill homogenizers (bead-beaters) on themarket including that disclosed in U.S. Pat. No. 5,567,050, entitled“Apparatus and method for rapidly oscillating specimen vessels”, whichdescribes an apparatus and method for rapidly oscillating specimencontaining vessels such as those used in an RNA recovery operationwherein small sized glass sized beads in the vessel are employed todisrupt the cell walls of an RNA component to release the RNA, includesa specimen vessel holder provided as a disc in which the containers arereceived. The disc is operably connected with oscillatory motionproducing means that in operation oscillates the disc rapidly in anoscillatory movement up and down symmetrically of a fixed vertical axis.The disc is haltered so it cannot rotate about the fixed axis. Lockingmeans in the form of a locking plate locks the vessels on the vesselholder and applies a clamping force thereto to prevent relative movementbetween the vessels and the holder to prevent generation of heat thatcould be of deleterious effect to the specimen material or the vesselsholding same.

Another example of a bead beater is described in U.S. Pat. ApplicationPublication No. 2012/0263010, entitled “Device for the Quick Vibrationof Tubes Containing, In Particular, Biological Samples” which disclosesan appliance for rapidly vibrating test tubes containing samples to beground up, the appliance comprising an electric motor for drivingrotation of a disk that is provided with an eccentric pin, the appliancebeing characterized in that the test tubes are perpendicular to theeccentric pin and are held by a clamp mounted on a support that issubstantially parallel to the eccentric pin, being connected to a fixedbaseplate via a Cardan type hinge having two mutually perpendicular axesof rotation (X and Y), one of which axes (Y) is substantially parallelto the eccentric pin and connects the support to the other axis (X) ofthe hinge that prevents the support from moving in rotation about aperpendicular axis (Z). The support is connected to the eccentric pin bya link.

Both of the above devices produce a figure 8 motion, with respectivecapacities of 12 x2mL and 3 x2mL sample tubes/vials, and peak to peakamplitudes of ⅝ and ¾ inch. Since the sample tubes/vials in bothexamples are 1.5 inches tall, this means that the sample and hardmatrices do not have the opportunity to travel throughout the wholelength of the sample vial and they stay mostly at the bottom of thesample vial. In contrast, the apparatus of the present inventionproduces an elliptical motion/path and has a peak to peak amplitudeequal to the length of the sample vial (1.5 inches, for example)allowing the matrices to fully interact with the sample throughout thesample tube/vial (sample processing chamber), making it much moreefficient, achieving the same results, as the devices above, in about⅒th the time of the other bead mill/bead-beater homogenizers.

Table 1 below outlines characteristics of commonly used sample grindersand homogenizers, for example, including a preferred embodiment of theapparatus of the present invention showing the peak to peak amplitudeadvantage of the present invention. The peak to peak amplitude of thepresent invention is not limited to 1.5 inches but is shown only as anexample. Peak to peak amplitude can be adjusted in the apparatus of thepresent invention to match the sample processing chamber (sample vial)length by varying the diameter of its crank to match the length of thesample processing chamber.

TABLE 1 Product Type Action Sample throughput Frequency Range (cpm)Amplitude (inches) Present Invention Bead-Beater crank-slider(combination of circular and linear motion) 1 750-4,400 1.5 XpeditionBead-Beater vertical linear impaction 1 3,600 0.25 Tissue-TearorRotor-stator probe rotor-stator 1 5,000-35,000 n/a Shredder SG3 Pressurehydraulic forcing of sample through a sieve with simultaneous rotationalmechanical grinding 1 200 n/a Disruptor Genie Bead-Beater horizontalorbital motion 12 1,000-3,000 0.5 Bead-Bug Bead-Beater Arc-shapedtrajectory (~2 inch radius) 3 2,700-4,000 0.75 Mini-Bead beater-1Bead-Beater Arc-shaped trajectory (~2 inch radius) 1 2,500-4,800 0.75Bullet Blender (BBX6F) Bead-Beater Horizontal linear impaction 24100-1,200 0.125 TissueLyserLT Bead-Beater Vertical linear motion 12900-3,000 0.75 PRECELLYS*24 Bead-Beater Arc-shaped trajectory (~3 inchradius) 24 6,500 0.315 MINILYS Bead-Beater Arc-shaped trajectory (~3inch radius) 3 3,000, 4,000, 5,000 0.63

SUMMARY

The present invention is an apparatus that generates a reciprocatingmotion for the purpose of grinding, or homogenizing (if liquid ispresent), biological or geological samples. A motor-driven crank islinked to a linearly-confined carriage via a connecting linkage. Thelinkage contains a holder for a sample tube or vial. The sample vial ispreferably cylindrical or rectangular in shape. As the crank rotates,the sample vial in the holder experiences an elliptical oscillatoryreciprocating motion that causes the sample and grinding media, such as,for example ceramic beads, within the sample vial to collide with eachother as they traverse the length of the sample vial. The sample breaksapart as a result of the collisions with the beads. The invention allowsfor substantial reduction of process time down to the range of 1 to 5seconds versus 5 to 60 seconds for bead-beaters commonly used bylaboratories, using the same reciprocation rates of about 4,000 to 5,000cycles per minute.

Compared to the above-mentioned bead-beaters, the apparatus of thepresent invention stands apart because it is more efficient andtherefore about 1 order of magnitude shorter processing times than otherbead-beaters. Its mechanism consists of a rotating crank connected to aslider via a connecting linkage (linkage). A holder for a sample vial ortube is attached to middle of the connecting linkage, which isapproximately 7.3 inches long in a preferred embodiment of the presentinvention but can be longer or shorter depending on the desiredelliptical path and the number of holders attached thereto, i.e., theapparatus of the present invention may include multiple holders. Thediameter of the crank is equal to or greater than the axial length ofthe sample vial (if the sample vial is 1.5 inches then the crankdiameter will be 1.5 inches or greater), so the crank diameterpreferably corresponds to the axial length of the sample vial or tube.For example, when a 1.5 inch sample vial is inserted into the holder, itwill undergo an elliptical oscillatory motion (elliptical path) withpeak to peak amplitude of 1.5 inches, causing the grinding matrices andsample within the sample vial or tube to traverse the whole length ofthe sample vial or tube thereby maximizing cascade impaction andshearing forces. With the other previously described bead-beaters, thematrices (beads) and samples tend to stay localized at the bottoms ofthe sample vial or tube, strongly influenced by gravity, which tends toseparate the heavy matrices from the lighter matrices into a densitygradient.

Another important characteristic that gives the apparatus of the presentinvention an advantage over the other bead-beaters is that the samplevial or tube in the apparatus of the present invention is rigidly heldat the top and bottom, making the collisions (during change of axialdirection) at those extremities mostly inelastic. This means that mostenergy will go into cascade collisions instead of moving the tube or thetube holder. The impaction-based bead-beaters experience inelasticcollisions mostly at the time of impaction. The rest of the time, thematrices and samples are colliding elastically within the tube. Thismakes processing with those bead-beaters much more inefficient andtime-consuming.

The present invention is specifically directed to a mechanicalreciprocating apparatus for grinding of samples comprising one or moresample vials or tubes of predetermined length, a grinding media in thesample vial, a holder to hold the sample vial, a connecting linkagewhere said holder is attached to, said connecting linkage having adistal and proximal end with pivot points at each end, a crank pivotconnected to the proximal end of said connecting linkage, a slide pivotconnected to the distal end of said connecting linkage, a frame top anda bottom frame wherein the various components of the apparatus areattached, a sliding carriage on a rail that is affixed to the frame top,said sliding carriage is connected to the slide pivot, a crank having adiameter equal to or greater than the length of the sample vial, saidcrank attached to the crank pivot; and a motor operatively connected tothe crank. The connecting linkage may also accommodate multiple holdersand the holders may be placed at various distances (adjustable) from theends of the connecting linkage to optimize its elliptical path ofsamples in the sample vial. In addition to varying the location of theholder/tube on the connecting linkage, the length of the connectinglinkage and the diameter of the crank may also be varied and optimizedto achieve an optimal sample/sample vial and/or media path to allowefficient grinding of the samples. The rail of the apparatus of thepresent invention may alternatively be attached to the connectinglinkage and the sliding carriage may be fixed to a pivot point on theframe top of the apparatus instead of the sliding carriage. Many typesof commercially available sample vials may be used in the presentinvention including, for example, but not limited to, cryogenic tubes,multi-well plates, plastic, metal or glass vials, and other sampleprocessing tubes/vials such as those listed in Table 2, below. Differenttypes of motors may also be used for the present invention including,for example; electrically, pneumatically, or hydraulically drivenmotors. The apparatus of the present invention may be configured as ahand held device or a table top device depending on its capacity forholding multiple sample vials or tubes.

TABLE 2 Model # Description Capacity (mL) Skirt Vendor 72.693 Screw captube 2 no Sarstedt 72.694 Screw cap tube 2 yes Sarstedt 72.730.406 Screwcap tube 0.5 yes Sarstedt 72.730.406 Screw cap tube 1.5 yes Sarstedt330TX Screw cap tube with extra thick walls 2 no Biospec 2007 Stainlesssteel tubes 2 no Biospec S6003-50 Lysis tubes prefilled with 2.0 mmdiameter ceramic balls 2 yes Zymo Research S6002-50 Lysis tubesprefilled with 0.5 mm diameter ceramic balls 2 yes Zymo Research

BRIEF DESCRIPTION OF THE DRAWINGS

Further objects of the present invention together with additionalfeatures contributing thereto and advantages accruing therefrom will beapparent from the following description of the preferred embodiments ofthe invention which are shown in the accompanying drawing figures withlike reference numerals indicating like components throughout wherein:

FIG. 1 is blow-up illustration of the apparatus of the present inventionshowing the individual components thereof;

FIG. 2 is an isometric view of the apparatus of the present invention;and

FIGS. 3A, 3B, 3C, and 3D are a top view of the apparatus of the presentinvention at four different angular positions of the crank.

DETAILED DESCRIPTION OF SOME EMBODIMENTS

The components of the preferred embodiment of the apparatus of thepresent invention are shown in the blown-up illustration of FIG. 1 . Theindividual components of the apparatus, with like reference numberscorresponding to the drawing of FIG. 1 , are listed below in Table 3:

TABLE 3 Ref No. Description 10 crank 11 crank pivot 12 sliding carriage/carriage/slide 13 slide pivot 14 rail 15 rail screws 16 linkage orconnecting linkage 18 holder 20 sample vial or tube 21 frame top 22ceramic bead 26 motor 28 drive belt 30 small pulley 32 big pulley 34spacer 36 spacer 38 big pulley shaft 40 small pulley shaft 42 framebottom 44 threaded adapter 46 frame screws 48 adapter screws 50 crankpivot bolt 52 slide pivot bolt 54 slide pivot nut 56 top frame screw 58slide pivot carriage screw 60 slide pivot bearing 62 crank pivot bearing100 reference line

As illustrated in FIG. 1 , a sample tube or vial (20) fits inside aholder (18). The holder (18) is attached to a connecting linkage orlinkage (16). The linkage (16) sits on top of bearing pivots; the crankpivot (11) and slide pivot (13). The proximal end of the linkage (16) isattached to the crank pivot (11) via the top frame screws (56) while itsdistal end is attached to the slider pivot bearing (60) in the sliderpivot (13) and is held in place via the slide pivot bolt (52) and slidepivot nut (54). The slide pivot (13) is connected to the slidingcarriage or slide (12) via slide pivot carriage screws (58). The slide(12) sits on top of the rail (14) which is attached to the frame top(21) via slide screws (15). The crank pivot (11) is attached to thecrank (10) via a crank pivot bearing (62). The crank (10) is connectedto the big pulley (32) via the big pulley shaft (38). The big pulley(32) is driven or turned via a small pulley (30) through a drive belt(28). The small pulley (30) is connected to a small pulley shaft (40)which connects directly to the motor (26). The big pulley (32), bigpulley shaft (38), drive belt (28) and the small pulley (30) are held inplace between the frame top (21) and frame bottom (42) via frame screws(46) and spacers (34 and 36). The frame bottom (42) is attached to athreaded motor adapter (44) via adapter screws (48). The threaded motoradapter (44) allows for the attachment of the device of the presentinvention to the motor (26) which drives the small pulley (30) therebymoving the sample vial (20) in an elliptical path at a predeterminedrate. In an alternate embodiment of the present invention, the slidingcarriage (12) and rail (14) is made longer so that the holder (18) couldbe placed directly on the sliding carriage (12). The motion of thesample vial (20) is linear in this alternative embodiment.

With reference now to FIG. 2 , there is depicted an isometric view of afully assembled apparatus of the present invention with the sample vial(20) in vial holder (18) which is in turn mounted on the connectinglinkage (16), which has a pivot point at each end for connecting it tosliding carriage (12) at the distal end and crank 10 at the proximal endas shown in FIG. 2 . Slide/carriage (12) slides on rail (14), which isfixed to the frame top (21) of the apparatus as discussed above. Motor(26) is linked to crank (10) via small pulley (30), drive belt (28), andbig pulley (32). As crank (10) is driven to rotate by motor (26), itsrotational motion is converted to linear motion as carriage (12) slideson rail (14). Since holder (18) is placed approximately halfway betweenthe crank (10) and carriage (12), it experiences a combination of linearand rotational motion, resulting in an elliptical trajectory of samplevial/tube (20).

The next figures, FIGS. 3A-D, show a top view of the apparatus at fourangular positions of the cycle of crank (10), as it rotates in acounterclockwise direction. Also illustrated is a ceramic bead (22) thatacts as pestle in grinding samples placed in the sample vial (20). Sincethe ceramic bead (22) has a high finite inertia, it will tend to stay inplace at the level of reference line (100) while vial (20) surroundingit reciprocates in an elliptical path.

Specifically, FIG. 3A depicts the ceramic bead (22) at midway the lengthof the vial (20), pressed against its left wall as it travels towardsthe top. FIG. 3B depicts the bead (22) impacting the top end of thetube. FIG. 3C depicts the ceramic bead (22) again midway the length ofthe tube, pressed against the right wall as it travels towards thebottom of the tube. Finally, the ceramic bead (22) impacts the bottom ofthe tube in FIG. 3D.

Operation

Example 1:

-   1. A sample to be ground or homogenized, if liquid is present, is    inserted into a sample vial (20) that has preloaded hard grinding    matrices [ceramic beads(22)] inside.-   2. The vial (20) is sealed and inserted into the holder (18) of the    device.-   3. The device is turned on for a set period of time (usually 5    seconds or less, for example) at about 4,000 to 5,000 cycles per    second to cause the sample to be ground or homogenized.-   4. The tube is removed from the holder and unsealed to remove the    ground sample for analysis.

Example 2:

-   1. The user determines the optimal location for the holder on the    connecting linkage.-   2. The holder is attached onto the connecting linkage at the    pre-determined optimal location (distance from the crank pivot) to    allow optimal grinding or homogenization of a sample.-   3. A sample to be ground or homogenized, if liquid is present, is    inserted into a sample vial (20) that has preloaded hard grinding    matrices [ceramic beads(22)] inside.-   4. The vial (20) is sealed and inserted into the holder (18) of the    device.-   5. The device is turned on for a set period of time (usually 5    seconds or less, for example) at about 4,000 to 5,000 cycles per    second to cause the sample to be ground or homogenized.-   6. The tube is removed from the holder and unsealed to remove the    ground sample for analysis.

Concluding Statements

All patents, provisional applications, patent applications and otherpublications mentioned in this specification are herein incorporated byreference.

While this invention has been described in detail with reference tocertain preferred embodiments, it should be appreciated that the presentinvention is not limited to those precise embodiments. Rather, in viewof the present disclosure, which describes the current best mode forpracticing the invention, many modifications and variations wouldpresent themselves to those of skill in the art without departing fromthe scope and spirit of this invention. The scope of the invention is,therefore, indicated by the following claims rather than by theforegoing description. All changes, modifications, and variations comingwithin the meaning and range of equivalency of the claims are to beconsidered within their scope.

Furthermore, those skilled in the art will recognize, or be able toascertain, using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

1. (canceled)
 2. An apparatus for the purpose of grinding orhomogenizing samples, the apparatus comprising: means for holding asample vial, the sample vial having a sample vial length; and means forgenerating reciprocating elliptical motion with a major axis that islarger than the sample vial length.
 3. The apparatus of claim 2, whereinthe means for generating elliptical motion includes a connecting linkagecoupled to a crank and a sliding carriage.
 4. The apparatus of claim 3,wherein the means for generating elliptical motion further includes amotor operatively coupled to the crank.
 5. The apparatus of claim 2,wherein the sample vial length is at least 1.5 inches.
 6. The apparatusof claim 2, wherein the means for holding the sample vial includes aconnecting linkage and a holder coupled to the connecting linkage. 7.The apparatus of claim 6, wherein the holder forms a cavity having acavity axial length that is greater than or equal to the sample viallength.
 8. The apparatus of claim 2, wherein the reciprocatingelliptical motion has a reciprocation rate of at least 4,000 cycles perminute.
 9. The apparatus of claim 8, wherein the reciprocation rate isconfigured to grind a sample or to homogenize a sample in 1 second orless.
 10. The apparatus of claim 2, wherein the means for holding thesample is further configured to hold a second sample vial parallel tothe sample vial.
 11. The apparatus of claim 10, wherein the secondsample vial has a second sample vial length that is less than or equalto the sample vial length.